Förster Resonance Energy Transfer (FRET) is used to visualize dynamic changes in protein interactions and activities within living cells. However, the spectral overlap between fluorescent protein (FP) donors and acceptors makes it challenging to accurately quantify changes in FRET signals and multi-color imaging of two or more FP FRET pairs in a single cell is extremely difficult. Lanthanide-based FRET (LRET) biosensors incorporate luminescent Tb(III) complexes with ms-scale excited-state lifetimes and multiple, narrow-line emission bands as donors and FPs (or other conventional fluorophores) as acceptors. Time-gated microscopy uses pulsed excitation and delayed detection to eliminate ns-scale fluorescence background and cleanly detect Tb(III)-sensitized FP emission. In order to increase LRET signals and achieve faster and more sensitive imaging, we have sought to develop biosensors that combine multiple Tb(III) donors with a single fluorescent acceptor. Due to the mismatch in lifetimes, multiple excited donors can successively transfer energy to one acceptor, thereby generating many photons with a single excitation.<br/>CPPs facilitate direct translocation of small molecules from culture medium into the cytoplasm. In order to inform our design of multi-Tb(III) probes, we conjugated oligoarginine to polyamidoamine (PAMAM) dendrons of different generations. We used FMOC-based, solid-phase peptide synthesis (SPPS) to prepare nonaarginine that was N-terminally coupled to PAMAM dendrons with 2, 4, and 8 branches (generations 0, 1 and 2, respectively). Each branch terminates in a primary amine which was coupled to 5,6-carboxyfluorescein. 2-dye and 4-dye Dendron conjugates entered the cytoplasm of HeLa cells within 30 min when cells were incubated at 4 °C or 37 °C in culture medium containing 5 µM or higher probe concentrations. The uptake of the 8-dye probe was not successful under any set of incubation conditions. We optimized the best condition for the probe delivery in which the preincubating of Hela cells with oleic acid can increase the direct transduction. Oleic acid can facilitate the delivery of a positively charged probe via electrostatic interaction. We can apply the final condition to deliver multiple-lanthanide probe into the cytoplasm to increase the probability of energy transfer from multiple donors to the fluorescence acceptor.

LRET imaging improvement with cell-permeable, multi-fluorophore protein labels

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.